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Molecular characterization of a family of metalloendopeptidases from the intestinal brush border of Haemonchus contortus

  • G. F. J. NEWLANDS (a1), P. J. SKUCE (a1), A. J. NISBET (a1), D. L. REDMOND (a1), S. K. SMITH (a1), D. PETTIT (a1) and W. D. SMITH (a1)...

Abstract

Substantial protection against the economically important parasitic nematode Haemonchus contortus has been achieved by immunizing sheep with a glycoprotein fraction isolated from the intestinal membranes of the worm (H-gal-GP). Previous studies showed that one of the major components of H-gal-GP is a family of at least 4 zinc metalloendopeptidases, designated MEPs 1–4. This paper describes aspects of the molecular architecture of this protease family, including the proteomic analysis of the MEP fraction of the H-gal-GP complex. These enzymes belong to the M13 zinc metalloendopeptidase family (EC 3.4.24.11), also known as neutral endopeptidases or neprilysins. The sequences of MEPs 1 and 3 suggested a typical Type II integral membrane protein structure, whilst MEPs 2 and 4 had putative cleavable signal peptides, typical of secreted proteins. Proteomic analysis of H-gal-GP indicated that the extracellular domain of all 4 MEPs had been cleaved close to the transmembrane region/signal peptide with additional cleavage sites mid-way along the polypeptide. MEP3 was present as a homo-dimer in H-gal-GP, whereas MEP1 or MEP2 formed hetero-dimers with MEP4. It was found that expression of MEP3 was confined to developing 4th-stage larvae and to adult worms, the stages of Haemonchus which feed on blood. MEP-like activity was detected in the H-gal-GP complex over a broad pH range (5–9). Since all 4 MEPs must share a similar microenvironment in the complex, this suggests that each might have a different substrate specificity.

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Tel: +44 (0) 131 445 5111. Fax: +44 (0) 131 445 6111. E-mail: george.newlands@moredun.ac.uk

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